PROJECT SUMMARY An emerging therapy for non-ischemic cardiomyopathy involves the delivery of human mesenchymal stem cells (hMSCs). Clinical trials document modest benefits on cardiac contractility, underscoring a need to better understand and exploit the underlying mechanisms governing hMSCs-cardiomyocyte (hCMs) interactome. Recent studies on hMSC-mediated heart therapies demonstrated that paracrine signaling?via secreted factors?is a crucial mediator of reduced cardiac fibrosis and enhanced angiogenesis. Moreover, hMSC paracrine factors have been shown to impact contractility by altering cardiomyocyte ion channel/pump activity. However, these findings fail to identify the key components of the hMSC secretome for enhancing contractility. We propose utilizing three-dimensional human engineered cardiac tissues (hECTs) as an in vitro model to investigate the role of hMSC exosomes in enhancement of cardiac contractility. Our lab recently discovered that hMSCs enhance hECT contractile force predominantly through paracrine signaling, counteracting adverse risks of hMSC-hCM heterocellular coupling. Importantly, we discovered that the exosomal component of the hMSC secretome is necessary and sufficient for hMSC-paracrine mediated enhancement of hECT contractility. Furthermore, by utilizing a systems biology approach and integrating hECT contractile function results with exosomal miRNA data, we predicted exosomal miRNA-21 as a lead candidate responsible for the favorable contractile effects of hMSC paracrine signaling. We later validated with qPCR that miRNA-21 levels are increased in hECTs supplemented with hMSC exosomes and hMSC total conditioned media relative to control, motivating our central hypothesis that exosomal miRNA-21 plays a key role in hMSC paracrine-mediated enhancement of human engineered cardiac tissue contractile performance. In testing this hypothesis, I will directly address an NHLBI topic of special interest (HL-142) by studying the role of exosomes as paracrine mediators in cardiovascular disease. In Aim 1, I will identify the role of exosomal miRNA-21 in hMSC-mediated enhancement of hECT contractility by: 1) treating hECTs with exosomes derived from hMSCs with miRNA-21 inhibitor (Sub-aim 1A); and 2) formulated lipidoid nanoparticle delivery of miRNA-21 mimic into hECTs (Sub-aim 1B). In Aim 2, I will test the role of hMSC exosomes on recovery of contractility using in vitro hECT models of acquired (Sub-aim 2A) and genetic (Sub-aim 2B) non- ischemic heart failure. Overall, the project is designed to frame the research within a clinical context, and provide a rigorous multi- disciplinary training in tissue engineering, systems biology, electrophysiology, stem cell biology, and biochemistry as a solid foundation on which to build my career as a future physician-scientist.